In vitro testing of salt coating of fabrics as a potential antiviral agent in reusable face masks

During the coronavirus disease (COVID-19) pandemic, wearing face masks in public spaces became mandatory in most countries. The risk of self-contamination when handling face masks, which was one of the earliest concerns, can be mitigated by adding antiviral coatings to the masks. In the present study, we evaluated the antiviral effectiveness of sodium chloride deposited on a fabric suitable for the manufacturing of reusable cloth masks using techniques adapted to the home environment. We tested eight coating conditions, involving both spraying and dipping methods and three salt dilutions. Influenza A H3N2 virus particles were incubated directly on the salt-coated materials, collected, and added to human 3D airway epithelial cultures. Live virus replication in the epithelia was quantified over time in collected apical washes. Relative to the non-coated material, salt deposits at or above 4.3 mg/cm2 markedly reduced viral replication. However, even for larger quantities of salt, the effectiveness of the coating remained dependent on the crystal size and distribution, which in turn depended on the coating technique. These findings confirm the suitability of salt coating as antiviral protection on cloth masks, but also emphasize that particular attention should be paid to the coating protocol when developing consumer solutions.

copies/mL) for 3 h at 34 °C in the presence of 5% CO2 under 100% relative humidity. After the incubation, unbound viruses were washed away by three rapid washes with the culture medium.
Residual viruses were collected by a 20-min apical wash and were quantified by quantitative reverse-transcription PCR (RT-qPCR; QuantiTect Probe RT-PCR; Qiagen, Hilden, Germany) to establish a baseline for viral replication at later time points. New virus particles were collected by 20-min apical washes at 24, 72, and 144 h post infection. Viruses produced over several days were pooled, quantified by RT-qPCR, aliquoted, and stored at -80 °C until use.

Scanning electron microscopy
Salt crystals on the fabrics were visualized by scanning electron microscopy (FEI Scios2; Thermo Fisher Scientific, Waltham, MA, USA) in low vacuum mode. The presence of salt impregnated in the fabric fibers was confirmed using energy-dispersive X-ray spectroscopy using an X-Max 50 mm 2 EDS Detector (Oxford Instruments, High Wycombe, UK) and the AZtec software.

MucilAir epithelium quality check
Airway epithelium is a layer of pseudostratified epithelium. When reconstituted on a semiporous membrane, epithelia have a homogenous and uniform appearance. As Transwell ® membranes (Corning, Glendale, AZ, USA) are clear and transparent, morphological changes in the cells grown on them can easily be monitored. Each insert was inspected under a conventional inverted microscope (Leica DMIRE2; Leica Microsystems CMS GmbH, Mannheim, Germany) to ensure the quality of the epithelia. The homogeneity and uniformity of the pseudostratified airway epithelia were inspected. Ciliary movement was clearly visible in all selected inserts, and the presence of mucus was detected by the refractive aspect of the apical surface.
All inserts used in the experiments were washed apically with culture medium 3 d before the experiments to remove accumulated mucus and cell debris and thus minimize the risk of interference with the toxicity tests. The transepithelial electrical resistance (TEER) was also measured to verify that all selected inserts satisfied the internal quality control standards (TEER > To check the integrity of the epithelia throughout the study, three selected inserts (mock) were not infected but exposed to 100 μL of culture medium on the apical side for 3 h and handled in the same way as the virus-exposed inserts. To one insert, Triton X-100 (100 μL of a 10% v/v solution in 0.9% saline) was added as a control of maximum epithelial disruption.

TEER measurement
For TEER measurements, 200 µL of buffered saline solution was added to the apical compartment of MucilAir cultures and resistance was measured using an EVOMX volt-ohm-meter (World Precision Instruments, Stevenage, UK). Resistance values (Ω) were converted to TEER values (Ω·cm 2 ) using the following formula: TEER (Ω·cm 2 ) = (resistance value (Ω) − 100(Ω)) × 0.33 (cm 2 ), where 100 Ω is the resistance of the membrane and 0.33 cm 2 is the total surface of the epithelium. as well as of controls for RNA extraction and RT-qPCR were included, and the reactions were run on a Chromo4 PCR Detection System from Bio-Rad (http://eqdb.nrf.ac.za/equipment-make/biorad; Hercules, CA, USA). The limit of quantification of the method was established at 390 gc/mL. Ct values were converted into RNA copy numbers per reaction using the slope-intercept form, corrected with the dilution factor applied during viral RNA extraction, and presented as log10 genome copy numbers per milliliter in graphs. Fig. S1. Energy-dispersive X-ray spectroscopy of spray and dip-coated fabric fibers. Spray treatments allowed the deposition of increasing amounts of salt by varying the valve aperture of the spray device according to arbitrary stroke units. (a) Spray, undiluted salt formulation, stroke unit 1 (Spr S1), (b) spray, 5-fold diluted salt formulation, stroke unit 3 (Spr S3 Dil5×), (c) spray, undiluted salt formulation, stroke unit 3 (Spr S3), (d) spray, undiluted salt formulation, stroke unit 5 (Spr S5), (e) spray, undiluted salt formulation, stroke unit 10 (Spr S10). For dip treatments test materials were immersed into (f) undiluted (Dip No Dil), (g) 5-fold diluted (Dip Dil5×), and (h) 10-fold diluted (Dip Dil10×) salt formulations.  area of the test sample analyzed by energy-dispersive X-ray spectroscopy.